Flávia Karina Delella

Finasteride Inhibits Human Prostate Cancer Cell Invasion through MMP2 and MMP9 Downregulation

Introduction The use of the 5-alpha reductase inhibitors (5-ARIs) finasteride and dutasteride for prostate cancer prevention is still under debate. The FDA recently concluded that the increased prevalence of high-grade tumors among 5-ARI-treated patients must not be neglected, and they decided to disallow the use of 5-ARIs for prostate cancer prevention. This study was conducted to verify the effects of finasteride on prostate cell migration and invasion and the related enzymes/proteins in normal human and tumoral prostatic cell lines. Materials and Methods RWPE-1, LNCaP, PC3 and DU145 cells were cultivated to 60% confluence and exposed for different periods to either 10 µM or 50 µM finasteride that was diluted in culture medium. The conditioned media were collected and concentrated, and MMP2 and MMP9 activities and TIMP-1 and TIMP-2 protein expression were determined. Cell viability, migration and invasion were analyzed, and the remaining cell extracts were submitted to androgen receptor (AR) detection by western blotting techniques. Experiments were carried out in triplicate. Results Cell viability was not significantly affected by finasteride exposure. Finasteride significantly downregulated MMP2 and MMP9 activities in RWPE-1 and PC3 cells and MMP2 in DU145 cells. TIMP-2 expression in RWPE-1 cells was upregulated after exposure. The cell invasion of all four tested cell lines was inhibited by exposure to 50 µM of finasteride, and migration inhibition only occurred for RWPE-1 and LNCaP cells. AR was expressed by LNCaP, RWPE-1 and PC3 cells. Conclusions Although the debate on the higher incidence of high-grade prostate cancer among 5-ARI-treated patients remains, our findings indicate that finasteride may attenuate tumor aggressiveness and invasion, which could vary depending on the androgen responsiveness of a patient’s prostate cells.

Fibronectin induces MMP2 expression in human prostate cancer cells.

High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the tumor cell lines commonly employed for prostate cancer research express only small amounts of MMPs when cultivated as monolayer cultures, in common culture media. The present study was conducted to ascertain whether culture conditions that include fibronectin can alter MMP2 and MMP9 expression by the human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2×10(4) cells/cm(2), cultivated until they reached 80% confluence, and then exposed for 4h to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 cells express detectable amounts of MMP9 when cultivated in common medium, whereas the addition of fibronectin induced high expression levels of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumor prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies of transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions.

Cytoxicity and Apoptotic Mechanism of Ruthenium(II) Amino Acid Complexes in Sarcoma-180 Tumor Cells

Over the past several decades, much attention has been focused on ruthenium complexes in antitumor therapy. Ruthenium is a transition metal that possesses several advantages for rational antitumor drug design and biological applications. In the present study, five ruthenium complexes containing amino acids were studied in vitro to determine their biological activity against sarcoma-180 tumor cells. The cytotoxicity of the complexes was evaluated by an MTT assay, and their mechanism of action was investigated. The results demonstrated that the five complexes inhibited the growth of the S180 tumor cell line, with IC50 values ranging from 22.53 µM to 50.18 µM, and showed low cytotoxicity against normal L929 fibroblast cells. Flow cytometric analysis revealed that the [Ru(gly)(bipy)(dppb)]PF6 complex (2) inhibited the growth of the tumor cells by inducing apoptosis, as evidenced by an increased number of Annexin V-positive cells and G0/G1 phase cell cycle arrest. Further investigation showed that complex 2 caused a loss of mitochondrial membrane potential; activated caspases 3, caspase-8, and caspase-9 and caused a change in the mRNA expression levels of caspase 3, caspase-9 as well as the bax genes. The levels of the pro-apoptotic Bcl-2 family protein Bak were increased. Thus, we demonstrated that ruthenium amino acid complexes are promising drugs against S180 tumor cells, and we recommend further investigations of their role as chemotherapeutic agents for sarcomas.

Caffeine reduces cadmium accumulation in the organism and enhances the levels of antioxidant protein expression in the epididymis

The aim of this study was to investigate the effects of caffeine (20 mg/L) intake on cadmium (15 mg/L) accumulation in the rat blood, testes, epididymis and prostate as well as cadmium-induced changes to the antioxidant defense system of the epididymis. Caffeine reduced the cadmium concentration in all tissues analyzed. Meanwhile, cadmium reduced catalase activity and increased superoxide dismutase (SOD) activity in the epididymis. Caffeine increased SOD activity, catalase and glutathione tissue expression and sustains the cadmiums effect on catalase and GSP-Px activity. No differences in the expression of metallothionein and lipid peroxidation were observed among the different treatments in the epididymis. In conclusion, low doses of cadmium alter the antioxidant enzymatic profile of the epididymis, but not induced oxidative lipid damage. Caffeine intake reduces overall cadmium accumulation in the organism and enhances the levels of antioxidant protein expression in the epididymis, thus exerting a protective effect against this metal.

Calcaneal tendon regions exhibit different MMP-2 activation after vertical jumping and treadmill running.

Increased activity of matrix metalloproteinases (MMPs) ‐2 and ‐9 was found in calcaneal tendon after physical training. However, little attention has been given to the distinct biomechanical and tissue structure of the calcaneal tendons proximal and distal regions. Herein, we evaluated the effect of two types of physical activities on tendon morphology and matrix metalloproteinase activities in the proximal and distal regions of rat calcaneal tendon, separately. Adult male Wistar rats from control, water‐adapted, vertical‐jumping, and treadmill‐running groups were sacrificed after 1 or 4 days of physical exercise, 6 hr after the end of that days exercise session. Tendons were processed for histology, morphometry, and gelatin zymography. Tendons from adapted and trained animals showed active secretory cells and increased thickness, cellularity, and blood vessel volume fraction of peritendinous sheath, but without inflammatory process. In the proximal region, both pro‐ and active MMP‐2 were increased after vertical jumping, but only pro‐MMP‐2 was increased after treadmill running. In contrast, in the distal region, both exercise types increased the activity of pro‐ and active MMP‐2, especially treadmill running, which increased the active MMP‐2 by about 11‐ and eightfold, respectively, after 1 and 4 days of training. No activity of MMP‐9 was observed in either tendon region in this study. In conclusion, distal and proximal regions of calcaneal tendon exhibit differential intensities of tissue remodeling after treadmill running or vertical jumping and MMP‐2, in the absence of inflammation, plays a major role in this adaptive response. Anat Rec, 2009.

Terminalia catappa L.: a medicinal plant from the Caribbean pharmacopeia with anti-Helicobacter pylori and antiulcer action in experimental rodent models.

ETHNOPHARMACOLOGICAL RELEVANCEnTerminalia catappa L. (Combretaceae) is a medicinal plant listed as a pharmacopeia vegetable from Caribbean to treat gastritis. The objective of this study was to evaluate the gastroprotective and healing effect of the aqueous fraction (FrAq) obtained from the leaves of Terminalia catappa and to determine the antiulcer mechanism of action in experimental rodent models and its activity to Helicobacter pylori.nnnMATERIAL AND METHODSnIn rodents, the FrAq was challenged by different necrotizing agents, such as absolute ethanol and ischemia-reperfusion injury. The antiulcer mechanism of action of FrAq was assessed and the healing effects of the fraction after seven and 14 days of treatment was evaluated by matrix metalloproteinase activity (MMP-2 and MMP-9). The toxicological effect of subacute treatment with FrAq during 14 days of treatment was also analyzed. The anti-Helicobacter pylori activity was determined by microdilution. The phytochemical study of the fraction was analyzed by experiments with FIA-ESI-IT-MS(n) (Direct Flow Analysis-ionization Electrospray Ion Trap Tandem Mass Spectrometry) and high performance liquid chromatography (HPLC) coupled to a photodiode array (PDA).nnnRESULTSnOral treatment with FrAq (25mg/kg) significantly decreased the number of ulcerative lesions induced by ethanol and ischemia/reperfusion injury. The action of FrAq was mediated by the activation of defensive mucosa-protective factors, such as increases in mucus production, the nitric oxide (NO) pathway and endogenous prostaglandins. Oral treatment with FrAq for seven and 14 days significantly reduced the lesion area (80% and 37%, respectively) compared to the negative control group. Analyses of MMP-9 and MMP-2 activity from gastric mucosa confirmed the accelerated gastric healing effect of FrAq. This extract also presented considerable activity against Helicobacter pylori. The mass spectrum and MS/MS of the aqueous fraction indicates the existence of many different phenolic compounds, including punicalagin, punicalin, and gallagic acid, among others.nnnCONCLUSIONSnWe concluded that FrAq from Terminalia catappa leaves has excellent preventive and curative effects on acute and chronic induced gastric ulcers and showed an important profile against Helicobacter pylori.

Finasteride treatment alters MMP-2 and -9 gene expression and activity in the rat ventral prostate.

The safety of using finasteride as a prevention of prostate cancer is still under debate. In this study, we investigated the effects of finasteride on the location, gene expression and activities of matrix metalloproteinases -2 and -9, which are involved in the degradation of extracellular matrix components during tissue remodelling and prostate cancer progression, invasion and metastasis. Ventral prostates (VP) from Wistar rats treated with finasteride (25 mg/kg/day) for 7 and 30 days and age-matched controls were evaluated using histology, immunohistochemistry, semi-quantitative RT-PCR and gelatin zymography. Finasteride treatment reduced the epithelial immunostaining of MMP-2 but increased MMP-9 immunostaining in the epithelial cells and in the stroma. The mRNA expression of both MMP-2 and MMP-9 were significantly increased on day 7 of finasteride treatment, mainly for MMP-9 and returned to the control levels by day 30. However, gelatin zymography showed that MMP-9 activity was significantly increased on day 7 of finasteride treatment and remained elevated on day 30 (p < 0.05), while MMP-2 activity was reduced after 30 days of treatment. Finasteride increases MMP-9 and reduces MMP-2 activities in the prostate, which may affect negatively and positively both normal and tumoural prostatic cell behaviour during the treatment. Studies on expression of MMPs in the prostate during different androgen manipulation or cancer chemoprevention strategies can contribute to understand the tissues overall response and clinical data.

Chronic caffeine intake increases androgenic stimuli, epithelial cell proliferation and hyperplasia in rat ventral prostate

Coffee intake has been associated with a low risk of developing cancer, including prostate cancer, which is one of the most commonly diagnosed cancer in men. However, few studies have evaluated the chronic effects of caffeine, which is the most abundant methylxanthine in coffee, on prostate morphology and physiology. In the present study, we investigated the effects of chronic, low‐dose caffeine intake on rat prostate morphology from puberty to adulthood. Five‐week‐old male Wistar rats were randomized into two experimental groups: caffeine‐treated (20 ppm in drinking water, n = 12) and control (n = 12). The ventral and dorsolateral prostates were dissected, weighted and submitted to morphological, morphometrical and immunohistochemical analysis of cellular proliferation, apoptosis and androgen receptor (AR) tissue expression. The testosterone (T) and dihydrotestosterone (DHT) concentrations were measured in the plasma. Our results show that caffeine intake increased the concentrations of T and DHT, organ weight, epithelial cell proliferation and AR tissue expression in the ventral prostatic lobe. All the ventral prostates from the caffeine‐treated animals presented various degrees of epithelial and stromal hyperplasia. Our results suggest that chronic caffeine intake from puberty increases androgenic signalling and cell proliferation in the rat prostate gland and can be related to the development of benign prostatic hyperplasia.

Metalloproteinase inhibition protects against reductions in circulating adrenomedullin during lead-induced acute hypertension

Intoxication with lead (Pb) results in increased blood pressure by mechanisms involving matrix metalloproteinases (MMPs). Recent findings have revealed that MMP type two (MMP‐2) seems to cleave vasoactive peptides. This study examined whether MMP‐2 and MMP‐9 levels/activities increase after acute intoxication with low lead concentrations and whether these changes were associated with increases in blood pressure and circulating endothelin‐1 or with reductions in circulating adrenomedullin and calcitonin gene‐related peptide (CGRP). Here, we expand previous findings and examine whether doxycycline (a MMPs inhibitor) affects these alterations. Wistar rats received intraperitoneally (i.p.) 1st dose 8 μg/100 g of lead (or sodium) acetate, a subsequent dose of 0.1 μg/100 g to cover daily loss and treatment with doxycycline (30 mg/kg/day) or water by gavage for 7 days. Similar whole‐blood lead levels (9 μg/dL) were found in lead‐exposed rats treated with either doxycycline or water. Lead‐induced increases in systolic blood pressure (from 143 ± 2 to 167 ± 3 mmHg) and gelatin zymography of plasma samples showed that lead increased MMP‐9 (but not MMP‐2) levels. Both lead‐induced increased MMP‐9 activity and hypertension were blunted by doxycycline. Doxycycline also prevented lead‐induced reductions in circulating adrenomedullin. No significant changes in plasma levels of endothelin‐1 or CGRP were found. Lead‐induced decreases in nitric oxide markers and antioxidant status were not prevented by doxycycline. In conclusion, acute lead exposure increases blood pressure and MMP‐9 activity, which were blunted by doxycycline. These findings suggest that MMP‐9 may contribute with lead‐induced hypertension by cleaving the vasodilatory peptide adrenomedullin, thereby inhibiting adrenomedullin‐dependent lowering of blood pressure.

Cadmium exposure inhibits MMP2 and MMP9 activities in the prostate and testis.

Matrix metalloproteinases (MMPs) are zinc (Zn(2+)) and calcium (Ca(2+)) dependant endopeptidases, capable of degradation of numerous components of the extracellular matrix. Cadmium (Cd(2+)) is a well known environmental contaminant which could impair the activity of MMPs. In this sense, this study was conducted to evaluate if Cd(2+) intake inhibits these endopeptidases activities at the rat prostate and testicles and if it directly inhibits the activity of MMP2 and MMP9 at gelatinolytic assays when present in the incubation buffer. To investigate this hypothesis, Wistar rats (5 weeks old), were given tap water (untreated, n = 9), or 15 ppm CdCl2 diluted in drinking water, during 10 weeks (n = 9) and 20 weeks (n = 9). The animals were euthanized and their ventral prostate, dorsal prostate, and testicles were removed. These tissue samples were processed for protein extraction and subjected to gelatin zymography evaluation. Additionally, we performed an experiment of gelatin zymography in which 5 μM or 2 mM cadmium chloride (CdCl2) was directly dissolved at the incubation buffer, using the prostatic tissue samples from untreated animals that exhibited the highest MMP2 and MMP9 activities in the previous experiment. We have found that CdCl2 intake in the drinking water led to the inhibition of 35% and 30% of MMP2 and MMP9 (p < 0.05) at the ventral prostate and testis, respectively, in Cd(2+) treated animals when compared to controls. Moreover, the activities of the referred enzymes were 80% and 100% inhibited by 5 μM and 2 mM of CdCl2, respectively, even in the presence of 10 mM of CaCl2 within the incubation buffer solution. These important findings demonstrate that environmental cadmium contamination may deregulate the natural balance in the extracellular matrix turnover, through MMPs downregulation, which could contribute to the toxic effects observed in prostatic and testicular tissue after its exposure.